Erythrocyte adhesion to polymer surfaces

Erythrocyte adhesion to polymer surfaces

Erythrocyte Adhesion to Polymer Surfaces 1 D. R. ABSOLOM,*,t'~'§ '2 W. ZINGG,*,§,II C. THOMSON,* Z. POLICOVA*'~" C. J. VAN OSS,~ AND A. W. NEUMANN*'t,...

824KB Sizes 0 Downloads 145 Views

Erythrocyte Adhesion to Polymer Surfaces 1 D. R. ABSOLOM,*,t'~'§ '2 W. ZINGG,*,§,II C. THOMSON,* Z. POLICOVA*'~" C. J. VAN OSS,~ AND A. W. NEUMANN*'t,§ *Research Institute, Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada; tDepartment of Mechanical Engineering, University of Toronto, Ontario M5S 1A4, Canada," C.Department of Microbiology, State University of New York, at Buffalo, New York 14214; §Institute of Biomedical Engineering, University of Toronto, Ontario M5S 1A4, Canada; and Illnstitute of Medical Science, University of Toronto, Ontario M5S 1A8, Canada Received June 22, 1983; accepted January 30, 1984 Thermodynamic model considerations suggest that the adhesion of biological cells to polymeric surfaces depends on the relative magnitude of the surface tension 3'sv of the substrates, the surface tension 3'cv of the cells, and the surface tension 3'LVof the suspending liquid medium. Glutaraldehydefixed erythrocytes are identified as suitable model particles for such studies. Experimental results of the extent of erythrocyte adhesion from suspension of mixtures of buffer and varying amounts of dimethyl sulfoxide (DMSO) confirm qualitatively and in certain aspects quantitatively the thermodynamic predictions. Specifically, the prediction that the extent of cell adhesion should become independent of substrate surface tension when 3'LV = 3~CVis confirmed by experiment. On the other hand for this case the free energy of adhesion predicting zero cell adhesion is not experimentally confirmed completely. However, performing these experiments with distilled water rather than buffer reduces erythrocyte adhesion to a negligible level. It emerges that van der Waals interactions are, by a large margin, the most important forces involved in cell adhesion to polymer surfaces. Nevertheless, not only divalent cations (through bridging effects), but also monovalent cations play a certain, though limited, role. © 1985 Academic Press, Inc.

In this paper, we have examined the role of these forces in determining the overall extent of erythrocyte adhesion to various polymer surfaces. In addition, we have investigated the influence of buffer composition, particularly in terms of ionic strength, on the residual level of cell adhesion where van der Waals forces are thought not to be operative.

INTRODUCTION

The adhesiveness of cells is a fundamental property of the cell surface. Cells can adhere to a variety of substrates such as glass (1-3), polymers (4, 5), metals (6), alkanes (7), lipids, and collagen (8). In recent years our studies have focussed on attempting to understand the fundamental mechanisms underlying the process of cell adhesion (9-13). The major forces of attraction between cells and substrate have been found to be the attractive van der Waals forces and plurivalent cation bridging (9, 10).

THEORETICAL CONSIDERATIONS

Our approach to the description of cell adhesion is based on surface thermodynamic considerations. Such an approach indicates that a properly identified thermodynamic potential, the grand canonical potential, which we simply call the free energy, will be minimized at equilibrium. This implies that the process under consideration, e.g., cell adhesion, will be favored if the process itself causes the thermodynamic function to de-

Presented at the symposium "Initial Events on Bioattachment at the Solid-Liquid Interface," held at the American Chemical SocietY meeting, Las Vegas, Nevada, March 1981, under the auspices of the Colloid Chemistry Division. ~,.~ 2 To whom correspondence should be addressed. 51

0021-9797/85 $3.00 Journal of Colloid and Interface Science, Vol. 104, No. 1, March 1985

Copyright © 1985 by Academic Press, Inc. All rights of reproduction in any form reserved.

52

A B S O L O M E T AL.

crease. The process will not be favored if it would cause the free energy function to increase. For a system in which the effect of electric charges as well as specific biochemical interactions (e.g., receptor-ligand) can be neglected, the net change in the free energy function is, per unit surface area, A F a d h = "YCS - - ~¢CL - - ~{SL

Ill

where 7cs is the cell-substrate interfacial tension, 7CL the cell-liquid interfacial tension, and 7SL the substrate-liquid interfacial tension. Although Eq. [1] by itself constitutes nothing more than a simple free energy balance, it is only in recent years that it has become possible to obtain experimental data for the various interracial tensions involving solid surfaces (14). Briefly, this is achieved through the use of Young's equation [2]

7SV - - 7SL ~--- '~LvCOS 0

where 7sv, 7SL, and 7LV are, respectively, the interfacial tension between a solid substrate S and the vapor phase V, between S and the liquid L, and between L and V; 0 represents the contact angle of the liquid on a solid (14). Of the four quantities in Eq. [2], only 7LV and 0 are readily determined experimentally; determination of a further relationship between these quantities is required. It has been shown previously from thermodynamic considerations that an equation of state relation of the form "YSL -~ f(Tsv,

')%v)

Through the use of Eq. [5] the unknown surface tension of the substrate, 7sv, may be determined from the measurable quantities 7LV and 0. If Eq. [4] is considered as a generic equation to calculate any interfacial tension, 712, from given (or predetermined) interfacial tension 7~3 and 723, where the subscripts 1, 2, and 3 refer to different phases, all the required interfacial tensions in Eq. [ 1] may be computed, thereby permitting explicit thermodynamic predictions of the relative extent of cell adhesion to various substrata with different surface properties. Equations [4] and [5] have, as they stand, certain purely mathematical limitations, which can, however, be circumvented (14, 16). Computer programs (11) as well as Tables (16) are also available which avoid such difficulties. The purpose of the present paper is to study the effect of the surface tension of the substrate (Tsv) and the suspending liquid medium (TLV)on the relative extent of adhesion of glutaraldehyde-fixed human erythrocytes to several polymers with a wide range of surface properties. A theoretical calculation of the net free energy change (AF adh) for the adhesion of erythrocytes from suspension to the various substrates as a function of 7sv is presented in Fig. 1 for two conditions o f the -12-

"/LV • "/CV

[3]

must exist (15). Using experimental contact angle data and liquid-vapor interfacial tensions, Eq. [3] has been formulated explicitly (14) as follows:

E u

~ &

0.015. ~ s v " 7LV"

[4]

In conjunction with Young's equation, this relationship follows: cos 0 = [0,0157sv - 2.00] ~f~sv. 7LV "~- 7LV 15 •

/vsv • ",,Lv -

l] [51

Journal of Colloid and Interface Science, Vol. 104, No. I, March 1985

-4-

% <1

7SL = 1

'

o

4

,o

3'0

;o

"

¢0

~/'SV [ergs/cm 2) FIG. 1. T h e free energy o f adhesion ( A F adh) for fixed h u m a n erythroeytes as a function of substrate surface tension, 7sv. (A) 3'Lv > 7 c v ; 7Lv = 72.8 e r g s / e m 2 a n d ~'cv = 64.6 ergs/cmZ; (B) 7LV < 7CV; 7LV = 59.8 ergs/ c m 2 a n d 7 c v = 64.6 e r g s / e m 2.

53

E R Y T H R O C Y T E ADHESION

liquid surface tension. The input data required for the development of such a plot are the respective surface tensions of the adhering cells ('~cv), the polymer substrates (~'sv) and the suspending liquid medium (3'LV)- This information is given in the legend to this figure. Consideration of such theoretical calculations as illustrated in Fig. 1 lead to a distinction between two situations: For YLV < "tcv

[6]

A F adh becomes more negative, i.e., decreases, with increasing (Tsv), predicting an increasing extent of cell adhesion with increasing substrate surface tension, 7sv, over a comparatively wide range of "rsv values. On the other hand, when "YLV > YCV

immediately that "~CL 0 and "Yes = "YSL. This limiting condition implies that cell adhesion, under these circumstances, does not depend on the surface tension of the substrates, and in principle should be zero if no other effects, such as electrostatic interactions, come into play (9, 13). In an earlier study, however, it was found that under these conditions a small but nevertheless positive level of leukocyte adhesion could be measured (10). Thus, the present study was expanded to investigate the influence of ionic strength and plurivalent cationic bridging on the overall extent of cell adhesion in an attempt to elucidate the nature of that residual level of cell adhesion. MATERIALS A N D M E T H O D S

[7]

the opposite pattern of behavior is predicted (9, 10). For the limiting case of the equality YLv = YCV

-~

[8]

becomes equal to zero independently of the value of 7sv. The latter fact can also be seen directly from the properties of the equation of state for interfacial tensions (13); since for the case of Eq. [8], it follows A F aan

Erythrocyte adhesion experiments were performed using the substrate materials listed in Table I with surface tensions ranging from 16 to 67 ergs/cm 2. Preparation of the surfaces was performed as described previously and as indicated in Table I (17, 18). The suspending liquid medium in these experiments was Hanks' balanced salt solution (HBSS), with different concentrations of dimethyl

TABLE I Solid Substrates Used in the Erythrocyte Adhesion Experiments

Preparation

Contact angle 0aao with water

Surface tension 3'sv (ergs/cm2)

Commercial Plastics, Toronto, Canada

Heat press

110 ___3

16.4

Dimethyldichlorosilane (SIL)

Eastman-Kodak, Rochester, N . Y .

Vapor deposition

108 + 2

17.6

Polystyrene (PS)

Central Research Lab., Dow Chemical Co.

Film

95 + 2

25.6

Low-density polyethylene (LDPE)

Commercial Plastics, Toronto, Canada

Heat press

84 + 4

32.5

Acetal resin (Ac)

Commercial Plastics, Toronto, Canada

Heat press

64 ___ 1

44.6

Sutfonated polystyrene (SPS)

Central Research Lab., Dow Chemical Co.

Film

24 _+ 3

66.7

Material Fluorinated ethylenepropylene

Copolymer (FEP)

Source

Journal of Colloid and Interface Sc/ence, Vot. 104, No. 1, March 1955

54

ABSOLOM ET AL.

sulfoxide (DMSO), up to a maximum of 18% (v/v), resulting in a range of liquid surface tensions from 72.8 to 59.8 ergs/cm2 (Table II). The pH was maintained at pH 7.1 through the addition of 0.01 M NaOH. The effect of ionic strength on the extent of adhesion was monitored through the use of various dilutions of HBSS and water. PREPARATION OF THE ERYTHROCYTE SUSPENSIONS

In order to clarify the mechanisms underlying the phenomena of cell adhesion, it was felt necessary to avoid the complications of living cells and physiological media from which complex macromolecules (e.g., proteins) would be expected to preadsorb onto the carefully prepared surfaces thereby making interpretation of experimental data more difficult. Thus, it was decided to employ glutaraldehyde-fixed human erythrocytes. Fixation results in the following advantages for in vitro adhesion experiments: It prevents the loss of proteins and glycoproteins from the cell surface; increases cell rigidity, thereby

TABLE II Suspending Media for the Erythrocyte Adhesion Experiments

Medium HBSS b HBSS-DMSO HBSS-DMSO HBSS-DMSO HBSS-DMSO HBSS-DMSO HBSS-DMSO HBSS-DMSO HBSS-DMSO H20-DMSO

1 3 5 10 12 12.5 15 18 12,5

DMSOa concentration (~ov/v)

Surface tension3'LV (ergs/cm2)

1 3 5 10 12 12.5 15 18 12.5

72.8 71.2 70,4 69.5 67.6 65.8 64.6 62.1 59.8 64.4

a Dimethyl sulfoxide. b Hanks' balanced salt solution, comprising in mg/liter: anhydr. CaCl2, 140.0; KCI, 400.0; KH2PO4, 60.0; MgC12.6H20, 100.0; MgSO4.7H20, 100.0; NaC1, 8000.0; NaHCO3, 350.0; NaHPO4.2H20, 60.0; glucose, 1000.0; ionic strength, # = 0.15; pH 7.26. Journal of Colloid and Interface Science, Vol. 104,No. 1, March 1985

stabilizing the area of contact between the cell and the substrate. Glutaraldehyde fixation (19) produces little change in the overall electrostatic charge on the cell surface (20) or in the local distribution of the chargebearing glycoproteins in the membrane lipid bilayer (21). After fixation and washing by centrifugation of the erythrocytes in the respective liquids (Table II), the cells were dialyzed against large volumes of the liquids for at least 2 hr to ensure that complete equilibrium had been achieved. Microscopic examination revealed that the erythrocytes were not clumped through DMSO incorporation. All the erythrocytes used in this work were from a single batch generously donated by Dr. R. S. Snyder of the Marshall Space Flight Center, NASA, Huntsville, Alabama. Adhesion of the erythrocytes to the various test surfaces was performed as described previously for leukocytes (9, 10), platelets (11), and bacteria (13). Briefly, I ml of cell suspension containing 1 × 106 cells in the appropriate liquid medium was placed on the surfaces and was retained in wells formed in Teflon molds separated from the polymers by Silastic gaskets. The cells were then incubated at 25°C for 30 min. Thereafter the surfaces were vigorously rinsed to remove nonadherent erythrocytes and the substrates were air dried. Then the number of cells adhering to the various surfaces was determined, after brief immersion in water to dissolve any salt crystals, using an automatic image analysis system (Omnicon 3000, Bausch and Lomb, Rochester, N. Y.) and expressed as the number of erythrocytes per unit surface area of substrate material. SURFACE TENSION DETERMINATIONS

Determination of the surface tension "Ysv of the solid substrates listed in Table 1 was performed by means of contact angle measurements, via an equation of state approach (9), as described above. Determination of the surface tension 3'uv

ERYTHROCYTE ADHESION of the various suspending liquid media was performed by means of the Wilhelmy plate method (22). The surface tension 3'cv of the glutaraldehyde-fixed human erythrocytes was determined by means of the freezing front technique which has been described in detail elsewhere (23). Briefly, the cells are placed in water which is then solidified in a controlled fashion. The interaction between the cells and the advancing water/ice interface is easily observed through a microscope (with a long working distance objective) with the aim of determining the critical velocity Vc of engulfment, i.e., the limiting velocity at which the particles are no longer pushed by the solidification front but, because of viscous drag, became engulfed in the solid phase. Dimensional analysis is used to relate the critical velocity to the free energy of adhesion for the particle at the advancing freezing front (24). The equation of state approach (14) thus provides the means for calculating the cell surface tension, "Ycv, from Vc measurements. RESULTS AND DISCUSSION In order to construct the predictive model (Fig. 1), it is necessary to know the surface tensions 3'LV of the suspending liquids, as well as the surface tension ~'cv of the fixed erythrocytes and the surface tension 3'sv of the various substrates. The surface tensions 3'sv were obtained from contact angle measurements and the values are listed in Table I. The liquid surface tensions 3'LV of the buffer and buffer-DMSO mixtures are summarized in Table II. The surface tension of fixed human erythrocytes was determined from the advancing solidification front experiments as described previously (23). This technique yields a temperature corrected surface tension value for the fixed erythrocytes of 64.9 ergs/cm2 at 22°C. This value agrees well with the surface tension of 65.0 ergs/ cm 2 at 22°C of the same batch of erythrocytes determined by an entirely independent method, viz., droplet sedimentation (25, 26).

55

It is relevant to note that all of the erythrocytes used in the work described in this report were from the same batch used previously in the freezing front and droplet sedimentation studies. As an illustration of the influence of the substrate surface tension on the extent of erythrocyte adhesion, we present in Fig. 2 photomicrographs of the substrate after rinsing. In Figs. 2a and b the liquid surface tension 3~LVwas 72.8 ergs/cm2 and the two substrates, fluorinated ethylene propylene copolymer (FEP) and sulfonated polystyrene (SPS), had surface tensions of 16.4 and 66.7 ergs/cm2, respectively. In Figs. 2c and d we show photomicrographs of the extent of adhering erythrocytes to the same two surfaces when the liquid surface tension had been substantially lowered to a value of 3'LV= 59.8 ergs/cm2. A comparison of Figs. 2a and b with Figs 2c and d reveals quite clearly that as the liquid surface tension is varied so the pattern of adhesion is changed. As the surface tension of the suspending liquid medium is lowered from 72.8 to 59.8 ergs/cm2, the number of adhering erythrocytes per unit surface area decreases in the case of FEP but increases for SPS under otherwise identical conditions. The effect of varying 3'LV on the extent of adhesion is perhaps even more clearly illustrated by considering one and the same substrate and two different ~'LVvalues, e.g., by comparing Fig. 2a with Fig. 2c or Fig. 2b with Fig. 2d. The results of the erythrocyte adhesion experiments from the various buffer/DMSO mixtures are summarized in Fig. 3. The theoretical predictions inherent in Fig. 1 and their implications are substantiated experimentally. At the lowest DMSO concentration, corresponding to the highest surface tension, YLV of the suspending medium, erythrocyte adhesion decreases with increasing substrate surface tension, 3'sv. As the DMSO concentration is increased and the surface tension 3'LV correspondingly lowered, the change in the extent of cell adhesion with increasing 3'sv becomes less pronounced. At a certain Journal of Colloid and Interface Science, VoL 104, No. 1, March 1985

56

ABSOLOM ET AL.

"Yt¥>Tcv

FEP

SPS

FIG. 2. Photomicrographs of erythrocyte adhesion under varying conditions. (a) Fluorinated ethylene propylene (FEP, 3'sv = 16.4 ergs/cm2); "rtv = 72.8 ergs/cm2. (b) Sulfonated polystyrene (SPS, 3'sv = 66.7 ergs/cm2); 3'LV = 72,8 ergs/cm2. (c) Fluorinated ethylene/propylene (FEP, ~/sv = 16.4 ergs/cm2); 3'LV = 59.8 ergs/cm2. (d) Sulfonated polystyrene (SPS, "rsv = 66.7 ergs/cm2); 3'LV = 59.8 ergs/em2.

t e n s i o n , "YLV, a d h e s i o n increases w i t h inc r e a s i n g 3'sv. A s i d e f r o m t h e i n t r i n s i c i n t e r e s t of these data and their possible implication

i n t e r m e d i a t e s u r f a c e t e n s i o n 3'LV, e r y t h r o c y t e a d h e s i o n b e c o m e s independent o f ~/sv a n d finally at y e t l o w e r v a l u e s o f t h e s u r f a c e

Liquid surface tensions'Ytv • = 72.8 ergs/cm2 v:71.2 . • ----70.4 ,, z, = 69.5 " -,=67.6 .

2000~ , ~ ~E

1600-

~

12o0-

{~].

o=65.8 s = 64.6

. .

w

5

800-

Z

400= ... ..=.~_~ ~ . . . . . o I

I

I

10

20

30

~

~_ I

40 'ysv(ergs/cm2)

~

!

.

I

I

I

50

60

70

FIG. 3. Erythrocyte adhesion as a function of substrate surface tension "Ysv for the various DMSO concentrations. Indicated error limits are 95% confidence limits (for graphical reasons error limits are given only for some cases; the errors are similar in all cases). Journal of Colloid and Interface Science, Vol. 104, No. 1, March 1985

57

ERYTHROCYTE ADHESION

in areas such as thromboresistance and blood previously suggested that it is reasonable to transfusions, there are two further points to assume a surface tension temperature depenbe made. First, the simple thermodynamic dence o f - 0 . 1 ergs/cm2/°C for biological model underlying Eq. [1], together with the materials (23). This contention is further equation of state approach for interfacial substantiated by the data presented in this tensions (14), describes the qualitative features paper. Correction of the data to 26°C, asof erythrocyte adhesion remarkably well. suming this temperature dependence factor, Second, the thermodynamic model predicts for the three techniques mentioned above that in the case "YLv= "YCV (Eq. [8]), A F adh (adhesion, freezing front, and droplet sedishould be independent of 3'sv, implying that mentation) yields values of ycv = 64.6, 64,5, the extent of adhesion should be independent and 64.3 ergs/cm2, respectively. of ~'sv, a situation that is indeed contained Thus, the thermodynamic model proposed in data of Fig. 3. To investigate this concept in Eq. [1] and illustrated in Fig. 1 can be further, the slopes of the straight lines in Fig. used to describe qualitatively erythrocyte 3 were plotted vs 3'LV in Fig. 4, by means of adhesion to a range of polymer surfaces a second order polynomial curve fit. It is under conditions of varying 3'LV- The model, inferred that the slope becomes equal to zero however, does not describe entirely all the at a value of 3'LV which is characteristic for features of the pattern of adhesion. At the the adhering species. This value, according 3'LV, where the extent of erythrocyte adhesion to the thermodynamic model, is equal to the is independent of the surface properties of surface tension of the erythrocytes. Consid- the substrate materials, eration of Fig. 4 reveals that for fixed human A F adh -- 0. [9] erythrocytes the adhesion slope becomes equal to zero when 3'LV = 64.6 ergs/cm2, Under these conditions (3/LV= ~/CV)however, implying that the surface tension of erythro- a small but nevertheless positive level of cytes is also equal to 64.6 ergs/cm2 at 25°C. adhesion to all of the surfaces was observed. This is in good agreement with the value of In this case, the adhesion presumably cannot ~'cv = 64.9 ergs/cm2 at 22°C obtained from be ascribed to a van der Waals attraction; in freezing front experiments (23) and ~cv a polar liquid such as water, electrostatic = 64.3 ergs/cm2 at 26°C obtained from drop- interactions may be implicated. To investigate let sedimentation studies (25, 26). We have this possibility further, the adhesion of eryth30

18-

6"5

o= o,

-6

-

-18

\

U)

-30

58

i

I

I

I

I

I

I

60

62

64

66

68

70

72

74

LIQUID VAPOUR SURFACE TENSION "/LV [erg s/cm2]

FIG. 4. Slopes of the straight lines of Fig. 3 vs ~'Lv. The slope is equal to zero for 3'LV = 3'cv. Journal of Colloid and Interface Science, Vol. 104, No. 1, March 1985

58

ABSOLOM ET AL.

rocytes to the various surfaces was measured at the liquid surface tension close to the cell surface tension of 64.6 ergs/cm 2. This was done at 12.5% DMSO at ionic strength ~t = 0.15 (in HBSS), i.e., just as in Fig. 3, and similarly at ~t = 0.075 (half-strength HBSS) and at # = 0. The results are given in Fig. 5. At ~t = 0.15, and in the absence of van der Waals interactions, an average of 104 adhering erythrocytes was found per square millimeter, while at/~ = 0.075 considerably fewer (-~68) erythrocytes per unit surface area are found. At ~t = 0 approximately only 6 cells per square millimeter were observed. Thus, lowering the ionic strength of the suspending medium significantly decreases the residual level of cell adhesion under these experimental conditions, i.e., when the van der Waals forces are reduced to zero. This reduction in the number of adhering cells may be due to electrokinetic phenomena such as a decrease in ionic strength being accompanied by an increase in ~'-potential. This possibility will be addressed in a subsequent publication, in which the results of an extensive study of erythrocyte adhesion to polymer surfaces and the influence of ionic strength and various anions/cations in solution will be described (28).

Finally, because plurivalent cations, e.g., Ca 2÷ ions, often are implicated in cell adhesion (29) and were present in our buffer system (HBSS) we also admixed a chelating agent, Na2EDTA, into the buffer in order to inactivate these cations. As indicated in Fig. 5, this indeed resulted in a significant decrease in the extent of cell adhesion to the polymer surfaces. The results of these experiments together with the data presented in Fig. 3 suggest that, in the absence of any specific biochemical interactions (e.g., receptor-ligand type bonds), two major types of forces play a role in determining the overall extent of cell adhesion to polymer surfaces: van der Waals attractive forces (between the adhering cells and the polymer substrate) and plurivalent cationic bridging. The data presented in this report would suggest that the most important of these forces are by a large margin the attractive van der Waals forces. When attractive van der Waals forces are operative, the level of erythrocyte adhesion out of HBSS onto a Teflon surface is ~ 1700 cells/mm 2. When the van der Waals attraction is reduced to zero, and under otherwise identical conditions, the level of cell adhesion is reduced to approximately 100 cells/ram 2 (~6%). Under these conditions both pluri-

100-

% T

T

--~-

t

l

.L

Ij. B

T

~ --_t

~2 U3

5O



HBsS + 12.5% D M S O



~ S t r e n g t h HBSS + 1 2 . 5 %

DMSO

,1, HBSS + 2.5% EDTA + 12.5% D M S O

Z



i I 10

± I 20

H20 + 12.5% D M S O

I

£

± I 30

I 40

±

5'0

go

7'0

")' s v ( e r g s / c m 2 )

FIG. 5. Erythrocyte adhesion as a function of substrate surface tension 3'sv in high and low ionic strength media and in the presenceof a chelatingagent. Indicated error limits are 95% confidencelimits. Journal of Colloid,and Interface,Science, Vol.

104, No. 1, M a r c h 1985

ERYTHROCYTE ADHESION valent cationic bridging a n d ionic strength c o n s i d e r a t i o n s a p p e a r to c o n t r i b u t e to t h e residual level o f cell a d h e s i o n . ACKNOWLEDGMENTS This work was supported in part by the Medical Research Council (MT5462, MA8024), the Natural Science and Engineering Research Council of Canada (A8278), and the Ontario Heart Foundation (4-12). One of the authors (D.R.A.) acknowledges support of the Ontario Heart Foundation through a Senior Fellowship. REFERENCES 1. Trommler, A., and Wolf, H., Stud. Biophys. 73, 223 (1978). 2. Mohandas, H., Hochmuth, R. M., and Spaeth, E. E., J, Biomed. Mater. Res. 8, 119 (1974). 3. Mayrovitz, H. N., Wiedeman, M. P., and Tuma, R. F., Thrombos. Haemostasis 38, 823 (1977). 4. Horisberger, M., Experentia 35, 612 (1979). 5. Horisberger, M., Physiol. Chem. Phys, 12, 195 (1980). 6. Gingell, D,, and Fornes, J. A., Biophys. 16, 1131 (1976). 7. Gingell, D., and Todd, I., J. Cell. Sci. 18, 227 (1975). 8. Smith, C. W., and Hollers, J. C., J. Clin. Invest. 65, 804 (1980). 9. Absolom, D. R., Neumann, A. W., Zingg, W., and van Oss, C. J., Trans. Amer. Soc. Artif. Intern. Organs 25, 152 (1979). 10. Absolom, D. R., van Oss, C. J., Genco, R. J., Francis, D. W., and Neumann, A. W., Cell Biophys. 2, 113 (1980). 11. Neumann, A. W., Hum, O. S., Francis, D. W., Zingg, W., and van Oss, C. J., J. Biomed. Mater. Res. 14, 499 (1980). 12. Neumann, A. W., Absolom, D. R., Zingg, W., van Oss, C. J., and Francis, D. W., in "Biocompatible Polymers, Metals, and Composites" (M. Syzcher, Ed.), Chap. 3, p. 53. Technomic Publishing House, Lancaster, Pa., 1983.

59

13. Absolom, D. R., Lamberti, F. V., Policova, Z., Zingg, W., van Oss, C. J., and Neumann, A. W., Appl. Environ. Microbiol. 46, 90 (1983). 14. Neumann, A. W., Good, R. J., Hope, C. J., and Sejpal, M., J. Colloid Interface Sci. 49, 291 (1974). 15. Ward, C. A., and Neumann, A. W., J. Colloid Interface Sci. 49, 286 (1974). 16. Neumann, A. W., Absolom, D. R., Francis, D. W., and van Oss, C. J., Sep. Purif. Methods 9, 62 (1980). 17. Chang, S. K., Hum, O. S., Moscarello, M. A., Neumann, A. W., Zingg, W., Leutheusser, H. J., and Ruegsegger, B., Med. Progr. Technol. 5, 57 (1977). 18. Omenyi, S., Ph.D. thesis, University of Toronto, 1978. 19. Sabatini, D. D., Bensch, K., and Barnett, R. J., J. CellBiol. 17, 19 (1963). 20. Pinto da Silva, P., J. Cell Biol. 53, 77 (1972). 21. Vassar, P. S., Hards, J. M., Brooks, D. E., Hargenberger, B., and Seamen, G. V. F., J. Cell Biol. 53, 809 (1972). 22. Neumann, A. W., Good, R. J., Ehrlich, P., Basu, P. K., and Johnston, G. J., J. Macromol. Sci. Phys. B7, 525 (1973). 23. Spelt, J. K., Absolom, D. R., Zingg, W., van Oss, C. J., and Neumann, A. W., CellBiophys. 4, 117 (1982). 24. Omenyi, S, N., Neumann, A. W., Martin, W. W., Lespinard, G. M., and Smith, R. P., J. Appl. Phys. 52, 796 (1981). 25. Omenyi, S. N., Snyder, R. S., van Oss, C. J., Ahsolom, D. R., and Neumann, A. W., J. Colloid Interface Sci. 81, 402 (1981). 26. Omenyi, S. N., Snyder, R. S., Absolom, D. R., van Oss, C. J., and Neumann, A. W., J. Dispersion Sci. Technol. 3, 307 (1982). 27. Neumann, A. W., Adv. Colloid Interface Sci. 4, 105 (1974). 28. Absolom, D. R., Neumann, A. W., Thomson, C., Policova, Z., and Zingg, W., Erythrocyte adhesion to polymer surfaces, II. In preparation. 29. Curtis, A. S. G., Biol. Rev. 37, 82 (1962).

Journal of Colloid and Interface Science, Vol. 104, No. 1, March 1985